1. Field of the Invention
This invention relates to methods and compositions for preventing or treating diseases of a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated. The effector molecule may be a molecule on a foreign organism. The foreign organism may be a virus.
The invention also relates to compositions, and to methods of identifying compositions, wherein the compositions are useful for treating mammalian diseases for which at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated.
The invention further relates to compositions and methods for targeting subject molecules to cells expressing DC-SIGN receptors, such as dendritic cells. These compositions and methods are based on targeting complexes, in which one or more subject molecules are covalently attached to one or more DC-SIGN blockers and, by virtue of binding of one or more of the DC-SIGN blockers of the targeting complex to DC-SIGN, the subject molecule is targeted to cells expressing DC-SIGN receptors.
2. Description of the Related Art
Dengue is an acute febrile tropical disease and the virus which causes it is an arbovirus which is transmitted by mosquitoes. The vectors of the disease are mosquitoes of the Aedes genus, in particular Aedes aegypti, which most commonly leave their larvae in domestic and peridomestic areas. The responsible virus, isolated in 1951, has been classified into four different antigenic types (DEN1, DEN2, DEN3 and DEN4). It belongs to the Flaviviridae family, genus flavivirus. 
More than two billion inhabitants live in endemic regions and the number of individuals infected by the virus is thought to be more than 100 million per year. Dengue is in particular responsible for 500 000 hospitalizations and for several tens of thousands of deaths annually, mostly children.
After an incubation of five to eight days, the clinical signs generally begin suddenly and consist of the appearance of undifferentiated fever (DF dengue fever) accompanied by severe headaches, lumbago, muscle and joint pain and also shivering. From the third to the fifth day of the febrile phase, a congestive maculopapular rash may appear for three to four days (conventional dengue).
In its severe form, the infection may result in the appearance of a hemorrhagic-syndrome (DHF or dengue hemorrhagic fever), characterized by increased vascular permeability and deregulation of hemostasis. Although, in the majority of cases, the disease generally evolves favorably within a week, it may turn out to be fatal in the event of hypovolemic shock (DSS or dengue shock syndrome). These complications may be due to the presence of preexisting immunity, acquired in particular during a primary infection with a heterologous dengue virus (different serotype). Specifically, two different types of serological response are identified in individuals infected with dengue: individuals who have never suffered a flavivirus infection and have not been vaccinated, against another flavivirus (yellow fever virus, Japanese encephalitis virus for example) will exhibit a primary response, characterized by a slow appearance of antibodies specific for the virus responsible for the infection; individuals who have already suffered a flavivirus infection (other dengue serotype for example) or have been vaccinated against another flavivirus will exhibit a secondary response, characterized by the rapid appearance of antibodies.
The infectious agent is the dengue virus which belongs to the Flaviviridae family, to which the yellow fever virus and the Japanese encephalitis virus also belong (T. P. Monath et al., (1996) Flaviviruses in B. N. Fields, D. M. Knipe, P. M. Howly et al. (eds.) “Fields Virology” Philadelphia: Lippincott Raven Press Publishers). These viruses have a single-strand RNA with positive polarity which comprises 11 000 nucleotides and which encodes a polyprotein of approximately 3400 amino acids. It is separated into three structural proteins and seven nonstructural proteins NS1, NS2A, NS28, NS3, NS4A, NS4B and NS5, during co-translational and post-translational cleavage by viral and cellular proteases. The NS1 nonstructural protein was identified for the first time in 1970 by P. K. Russel et al. (J. Immunol., (1970), 105, 838-845) and characterized in 1985 by G. W. Smith et al. (J. Gen Virol., (1985), 66, 559-571). This glycoprotein, which is highly conserved in the flavivirus genus (T. P. Monath already mentioned), in particular in the four dengue virus serotypes, exists in an intracellular form and in an extracellular form. The intracellular form is thought to be involved in the early phases of replication of the virus (Hall R. A. et al., J. Virol. (1999), 73, 10272-10280; Rice C. M. at al., J. Virol., (1997), 71, 291-298; Rice C. M. et al., J. Virol., (1996), 222, 159-168; Rice C. M. et al., J. Virol., (1997), 71, 9608-9617). Before being transported to the plasma membrane, the NS1 protein undergoes dimerization. In mammalian cells, but not in insect cells, a portion of the NS1 protein is released into the extracellular medium, either primarily in the form of a soluble protein, or secondarily in a microparticulate form. When it is in a soluble form, the protein exists in the form of an oligomer, in particular of a pentamer or of a hexamer (Crooks A. J. et al. J. Chrom. (1990), 502, 59-68 and J. Gen. Virol. (1994), 75, 3453-3460 and Glamand et al., J. Virol. (1999), 73, 6106-6110).
No specific treatment exists and the care given to the patient is uniquely symptomatic. In the case of conventional dengue, the treatment is based on the administration of analgesics and antipyretics. In the case of DHF, the treatment consists of an infusion to compensate for the plasma leakage, combined with correction of hydroelectric problems and reinitiation of diuresis.
There is no commercially available vaccine against the dengue virus. On the other hand, protection assays with attenuated strains of the 4 dengue virus serotypes have been carried out by N. Bhamarapravati et: al. (Dengue and Dengue haemorrhagic fever (1997), 367-377), with unsatisfactory results. Prevention is therefore based solely on combating the vector. This combat combines larval destruction and “adulticide” spraying.
The pathogenesis of severe forms of dengue (DEN) virus infection is not completely understood. Substantial T cell activation is observed in severe DEN disease. A number of cytokines and chemokines are found to be elevated in dengue hemorrhagic fever and/or dengue shock syndrome. Macrophages have long been recognized as an important component of DEN pathogenesis. As emphasized by Palucka, immature human dendritic cells (DC), in contrast to other leukocytes, were preferentially permissive for DEN infection (Wu et al., Nat. Med. 6:816, 2000; for review: Palucka, Nat. Med 6: 748, 2000). Unlike monocytes/macrophages, DEN virus infection was not enhanced by specific antibody (Marovich et al., JID Symp. Proc. 6:219, 2001). Immature DCs can undergo maturation in response to DEN virus infection. Upregulation of surface markers B7-1, B7-2, HLA-DR CD11b and DC83 and cytokine production were observed following infection of DCs. There is growing evidence that DEN infection can induce functional maturity in DC. Such infection causes upregulation of surface markers B7-1, B7-2, HLA-DR CD11b and DC83 and stimulates cytokine production (Ho et al., Immunology 166: 1499, 2001). Immature DCs exposed to DEN virus produce TNF-α, which may perturb endothelial cell function.
Dendritic cells (DC) are specialized Antigen Presenting Cells (APC) involved in the initiation of T cell-dependent immune responses as consequence of their high expression of MHC and costimulatory molecules. Myeloid DC are distributed throughout the body in an immature state, exhibiting a high capacity for antigen uptake and processing. Once activated by inflammatory stimuli or infectious agents, DC undergo a maturation process, migrate to lymphoid organs, and acquire their capacity to activate naive T lymphocytes.
An important question is which of DC-specific molecules DEN virus uses as a receptor for entry. The human DC-specific adhesion receptor DC-SIGN (ICAM-grabbing non integrin or CD-209), a type 11 integral protein, is of particular interest because its expression is largely restricted to immature DCs. DC-SIGN has been shown to be the ligand of ICAM-3, which enables transient DC-T cell interactions, thus facilitating primary immune response (Geijtenbeek et al. Nature 1: 353, 2000). DC-SIGN appeared to be a critical mediator of the migratory and T cell-interacting capabilities exhibited by maturing immature myeloid monocyte-derived DCs. DC-SIGN expression is IL-4 dependent and is negatively regulated by IFN-γ, IFN-γ, TGF-β, and anti-inflammatory agents (Relloso et al., J. Immunol. 168: 2634, 2002). DC-SIGN polymorphisms might also explain why some patients mount protective immunity whereas other do not.
DC-SIGN, a C-type lectin, has a single carbohydrate recognition domain that interacts with proteins with either mannose or galactose side chains in a calcium-dependent manner (Drickammer, Curr. Opin. Immunol. 13: 585, 1999). DC-SIGN is now believed to bind to high-mannose oligosaccharides that are present on the viral glycoproteins and thereby may capture enveloped viruses (Feinberg et al., Science 294: 2163, 2001). For example, DC-SIGN binds to the HIV envelope glycoprotein gp120 (Geijtenbeek et al. Cell, 100: 587, 2000) and thereby mediates rapid internalization of intact HIV into a nonlysosomal compartment (Kwon et al., Immunity, 16: 135, 2002).
There exists a need in the art to develop methods and compositions for modulating the specific binding of effector molecules to the DC-SIGN receptor, for example on the dendritic cells of mammals. Such methods and compositions are needed, for example, to prevent and treat diseases such as viral infections; for example Dengue virus infections. In this regard, there is a need to identify cell proteins involved in viral attachment and/or fusion. Additionally, methods and compositions are needed that allow the specific targeting of cells expressing DC-SIGN receptor, such as dendritic cells or alveolar macrophages, to aid in therapy or diagnosis.